1. Field of the Invention
The present invention relates to two-photon fluorescent probes for imaging acidic vesicles in live cells and tissue and a method for imaging acidic vesicles in live cells and tissue using the two-photon fluorescent probes. More particularly, the present invention relates to two-photon fluorescent probes that have a large penetration depth and are selectively and clearly capable of visualizing vesicles under acidic conditions, and a method for imaging acidic vesicles in live cells and tissue using the two-photon fluorescent probes.
2. Description of the Related Art
Lysosomes and lysosome-related organelles constitute a system of acidic compartments (pH 4.0-5.0), which contain a large number of enzymes and secretory proteins exhibiting a variety of functions. To determine their functions, a variety of membrane-permeable fluorescent pH and lysosomal probes have been developed with some of them being commercially available.
However, use of these probes with one-photon microscopy (OPM) requires excitation with short-wavelength light (˜350-550 nm) and presents several problems such as shallow penetration depth (<80 μm), photobleaching and cellular autofluorescence.
As an alternative method to solve the problems associated with the use of OPM, the use of two-photon microscopy (TPM) is considered.
TPM employs two near-infrared (NIR) photons for excitation and is of particular interest in tissue imaging (W. Denk, J. H. Strickler, W. W. Webb, Science, 1990, 248, 73.; W. R. Zipfel; R. M. Williams; W. W. Webb, Nat. Biotechnol. 2003, 21, 1369).
In 1932, Maria Goeppert-Mayer predicted a phenomenon in which two photons are spontaneously absorbed (Ann, Phys. 9 (1931) 273), but this two-photon excitation had not been practically utilized until strong laser sources were developed. Two-photon excitation is referred to as a phenomenon in which two photons are simultaneously absorbed in the same fluorophore having a sufficiently high photon density per unit volume and time by irradiation with a strong light source. The absorbed energy is the same as the sum of the energies of the two photons and the possibility of two-photon excitation is dependent on the square of the photon density.
Accordingly, the absorption of two photons is a secondary non-linear optical phenomenon. The photons in the excited state transit to the ground state and emit energy as fluorescence corresponding to the bandgap energy. This energy emission is called ‘two-photon fluorescence’. It should be understood that the emitted photonic energy is greater than the photonic energy of an irradiation source. Substances emitting fluorescence by two-photon excitation are commonly termed ‘two-photon probes’. Such two-photon probes may be excited by means of a light source capable of providing photonic energy corresponding to the bandgap energy. This excitation is referred to as ‘one-photon excitation’. A fluorescence emission spectrum obtained by two-photon excitation has the same spectral properties as that obtained by one-photon excitation.
A first characteristic of two-photon excitation is that the excitation occurs only near the limited three-dimensional regions of light irradiators, and therefore, fluorescence emission obtained by the excitation is localized in three-dimensional space, resulting in a minimization of background fluorescence. A second characteristic of two-photon excitation is that the wavelength of the irradiated light is different from that of the emitted fluorescence. Particularly, the two-photon excitation is useful in observing small-volume samples because the excitation volume is very small.
Based on the above-mentioned characteristics, two-photon microscopy capable of inducing two-photon excitation by irradiation of light in the near-infrared region is currently in the spotlight in bioimaging applications. The reason for this is due to the following advantages: i) little damage of biomolecules by irradiation of near-infrared light, which enables the application of two-photon microscopy to living cells; ii) large penetration depth of near-infrared light; and iii) minimized tissue auto-fluorescence. Two-photon probes used for two-photon microscopy must satisfy the following requirements: i) large two-photon cross section (δTPA) in the near-infrared region; ii) suitable water solubility, iii) high photostability; and iv) high binding selectivity for live cells and tissue.
However, most of fluorescent markers (two-photon fluorescent probes) presently used for TPM have small two-photon action cross sections (Φδ) that limit their usage. Particularly, acidic conditions found in living cells and tissues extremely limit the efficiency of the conventional two-photon fluorescent probes. Two-photon fluorescent probes as effective markers that can visualize vesicles under acidic conditions have never been, to our knowledge, reported or developed.
An ideal two-photon fluorescent probe for staining acidic vesicles in cytosol selectively permeates the cytosol and stains vesicles without staining membranes dividing the cytosol to emit fluorescence. However, conventional two-photon fluorescent probes stain membranes as well as cytosol to cause the problem of mistargeting. Under these circumstances, there is an urgent need to develop two-photon fluorescent probes that can selectively stain vesicles in cytosol under acidic conditions to visualize the vesicles.